Qz. Zhu et al., APPLICATION OF HEMIN AS A LABELING REAGENT IN MIMETIC ENZYME-IMMUNOASSAY FOR HEPATITIS-B SURFACE-ANTIGEN, Analytical letters, 31(6), 1998, pp. 963-971
A novel mimetic enzyme immunoassay method has been developed for the d
etermination of hepatitis B surface antigen (HBsAg) in solution. Hemin
, a horseradish peroxidase substitute, was used as a labeling reagent
to catalyze the reaction of p-hydroxyphenyl acetic (HPA) and hydrogen
peroxide in alkaline medium. In the sandwich immunoassay, anti-HBsAg a
ntibody was coated on a 96-well plate (polystyrene), which first react
ed with standard HBsAg solution or HBsAg in the test blood serum and t
hen further reacted with the fixed amount of hemin-labelled anti-HbsAg
. After the two-step immunoreaction, the immunochemically adsorbed hem
in-anti-HBsAg conjugate moiety was determined by measuring the fluores
cence produced in a solution containing HPA and hydrogen peroxide. The
fluorescence intensity was directly proportional to the concentration
of HBsAg. The calibration graph for HBsAg was linear over the range 0
-450 ng/well with a detection limit of 0.9 ng/well. The method is sens
itive enough for the determination of HBsAg level in hepatitis B blood
serum.